Cross-linking mass spectrometry and structural modeling identifies compact conformation of DENV NS2B cofactor region...

Dengue virus infection remains a public health threat. Dengue NS2B-NS3 proteins are prime antiviral drug targets, highly dynamic, and adopt different structural conformations. We combine cross-linking mass spectrometry (XL-MS), molecular dynamics (MD) simulations, and biochemical assays to identify NS2B-NS3 full length interactions. Using cross-linkers of different lengths as molecular rulers, we identified NS2B S48 as a key interacting residue with NS3 by XL-MS. Structural modeling with MD simulations revealed a novel compact conformation of the NS2B-NS3 complex. Mutation of NS2B S48 to alanine or lysine greatly reduced protease activity and disrupted the binding pocket in MD simulations with a loss of NS2B-NS3 interactions. Additionally, NS2B-NS3 cross-links were found to be conserved across all four dengue serotypes. Our interdisciplinary approach reveals a new key interacting residue and a compact conformation that are structurally and functionally important for the dynamic NS2B-NS3 complex. These results can help guide drug development against dengue.